Human tryptophan dioxygenase (hTDO) is an important hemeprotein that catalyzes the initial and rate-limiting step of the kynurenine (KYN) pathway?the conversion of Trp to N-formyl kynurenine (NFK). It is well-accepted that hTDO plays a critical role in controlling Trp homeostasis in liver. More recently, it was discovered that hTDO is also expressed in a variety of human tumors to suppress antitumor immunity, thereby promoting aggressive tumor growth. In addition to cancer, hTDO has been implicated in a wide spectrum of neurodegenerative diseases, such as Alzheimer?s and Huntington?s diseases, as well as depression. hTDO has hence been recognized as an important therapeutic target. Despite its importance, the progress has been limited by the poor knowledge of the molecular properties of the enzyme. Recently, we have made several major breakthroughs in (i) developing a new protocol allowing the preparation of high quality hTDO protein, allowing the documentation of the first glimpse of the structural features critical for the dioxygenase chemistry, as well as an unusual activity of the ferric enzyme, (ii) solving the first substrate-bound 3D-structure of hTDO, as well as trapping reaction intermediates in single crystals and solved their structures, which unveiled several previously unidentified structural features that are critical for controlling the catalytic activity and cellular degradation of the enzyme, one of the most rapidly degraded enzymes in liver, and (iii) obtaining the first evidence supporting the cellular degradation of hTDO via a ubiquitination-linked proteasomal degradation pathway. These important achievements allow us to build novel hypotheses and propose a comprehensive approach from a unique perspective to test them. The main objective of this project is to delineate the structure, function and regulation of hTDO, as part of our long term goal in comprehending heme dioxygenase chemistry and TDO biology. The specific aims of our project are: (i) Identify the novel dioxygenase intermediates of hTDO. (ii) Determine the dioxygen activation mechanism of the unique ferric reaction. (iii) Delineate the product release mechanism of hTDO. (iv) Delineate the exosite-dependent regulation mechanisms of hTDO. We anticipate that fulfilling the objectives of this project will result in new insights into the structure, function and regulation of this essential enzyme, and provide an important blueprint for the design of hTDO-selective inhibitors as therapeutics for cancer, neurodegenerative diseases and depression.